 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 29, 2018 |
| Title |
BMCP_75 |
| Sample type |
SRA |
| |
|
| Source name |
Bone marrow cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell population: BMCP
sorting strategy: Live Lin- cKit+ Sca1- integrin beta7hi CD16/32hi cells
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cells were sorted into 96 well PCR plates containing 2.3 µl lysis buffer consisting of 2.3 U SUPERase In™ RNase Inhibitor (ThermoFisher Scientific, AM2696) in 0.2 % Triton per well. Cells were processed for RNA-sequencing following Picelli et al (Nature Protocols 9, 171-181 (2014)). Briefly, oligo-dT was annealed to polyA-mRNA at 72 °C for 3 minutes, followed by cDNA synthesis using SuperScript™ II Reverse Transcriptase (Invitrogen 18064014). PCR was performed with 23 PCR cycles and products cleaned up using 1:0.6 ratio of cDNA: AMPure XP beads (Beckman Coulter). ERCC Spike In Mix was included in each single cell reaction at 1: 3 000 000 dilution (ThermoFisher Scientific 4456740). Single cell cDNA library was eluted in 20 µl volume of EB Buffer (Qiagen).
Prior to library preparation, cDNA library was checked on Agilent Bioanalyser 2100 with Agilent High Sensitivity DNA Kit. Library preparation was performed using Illumina Nextera XT DNA Library Preparation Kit with 1.26 µl cDNA library and Nextera XT Index Kit v2 Set A and D. Single cell libraries were pooled for all indexed samples and cleaned with AMPure XP beads; first with 1:0.5 ratio of cDNA: beads then the supernatant cleaned with 1:0.2 ratio of cDNA: beads. Pooled libraries were quantified with KAPA Library Quant Kit (KAPA Biosystems KK4824) and single-end 50 base pair sequencing was performed on the Illumina HiSeq 4000 platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
RNA-seq reads were aligned to mm10 using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1).
Reads in features were counted with htseq-count (HTSeq version 0.5.3p3) with the parameter (-s no).
Genome_build: mm10
Supplementary_files_format_and_content: HT-Seq counts(.txt)
Supplementary_files_format_and_content: extra_sequences.fa: Fasta file contains the sequences used for the customized index.
|
| |
|
| Submission date |
Nov 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Evangelia Diamanti |
| E-mail(s) |
ed347@cam.ac.uk
|
| Phone |
01223 62317
|
| Organization name |
University of Cambridge
|
| Department |
Haematology
|
| Lab |
Gottgens
|
| Street address |
Wellcome Trust / MRC Building, Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE106973 |
Single cell RNA sequencing of murine basophil/mast cell progenitors and granulocyte/monocyte progenitors |
|
| Relations |
| BioSample |
SAMN08031845 |
| SRA |
SRX3398210 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
